Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing

Abstract

BackgroundThe transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening.MethodsWe developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS.ResultsWe identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %.ConclusionThese results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2004-4) contains supplementary material, which is available to authorized users.

Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum

“…Mutations that occurred more than once per animal were considered to have originated from a single independent event via clonal expansion. Using the limit of detection/linear model16 to correct for clonal expansion we identified 427 independent mutations (131 in controls and 296 in the BaP-exposed group). As shown in Table 1, the average clonality in the germ cells of BaP-exposed animals (24.8%) was lower than in control animals (41%).…”

“…No silent mutations were observed in controls, while they represented 5.6% of the mutations induced by BaP (but this was not significantly higher than controls). These silent mutations likely represent “hitch-hiker” mutations that co-occurred with other mutations that were detectable by the positive selection assay16.…”

“…We compared the control and BaP-induced mutation spectra in sperm to the control and induced spectra in bone marrow published previously16 (Fig. 3 and Table 3).…”

“…Previously, sequencing data were limited to very low copy numbers of the smaller cII transgene due to technical restrictions. However, when coupled with next generation sequencing (NGS) technologies, the TGR assay can be used to generate a high-resolution mutation spectrum of the larger lacZ transgene, providing coverage of a much greater number of mutants1617. Thus, NGS- lacZ analysis allows the detection of subtle differences in mutation spectra that may otherwise be missed when analyzing a smaller sample of mutants.…”

“…Here, we use the TGR mutation assay coupled with NGS to produce high-resolution spectra of mutation types in the germ cells of untreated and BaP-exposed male mice. We then compare these to the spontaneous and BaP-induced mutation spectra in bone marrow16 and speculate on the possible mechanisms responsible for any spectral discrepancies between these tissues. This study provides important evidence for elucidating whether or not there are germ cell-specific mechanisms for spontaneous and BaP-induced mutations.…”